FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
Title of the invention
A NOVEL PROCESS FOR SYNTHESIS OF 37 MER PEPTIDE
Applicant(s)
Name Nationality Address
USV LIMITED Indian company incorporated BSD MARG, ARV1ND VITTHAL GANDHI CHOWK,
under Companies Act, 1956 GOVANDI (E), MUMBA1400 088
3. Preamble to the description
The following specification particularly describes the invention and the manner in which it is to be performed.

TECHNICAL FIELD:
The invention relates to a novel solid phase peptide synthesis of a 37-mer amyloid peptide, Pramlintide, and intermediates. More particularly the invention relates to the surprising finding of use of a single pseudoproline moiety for the synthesis of Pramlintide giving high purity as compared to the use of more than one Pseudoproline moieties. Further the invention also relates to a surprising finding of improvement in purity of the crude peptide by modifying the deprotection solvent system in addition to the incorporation of a single pseudoproline moiety. The instant invention also focuses on use of relatively optimally linker loaded acid labile resins for cost effective and efficient synthesis of pramlintide.
BACKGROUND AND PRIOR ART
Solid phase peptide synthesis is an alternative to solution phase synthesis. Currently large number of peptides have been successfully produced on a large scale using solid phase peptide synthesis.
Despite having advantages over solution phase synthesis, sometimes the synthesis process becomes highly expensive due to the heavy cost incurred by raw materials used for the synthesis for e.g. , protected amino acids, amino acid derivatives and specific resins. The use of expensive amino acid derivatives becomes a major setback in the synthesis, as the process becomes very expensive due to which the commercial scale production of the peptide hinders affecting the commercial business gain.

US5686411 discloses the solid phase symthesis of amylin and amylin analogs using methylbenzhydrylamine anchor-based resin and Na-Boc/benzyl-side chain protection. Also it discloses the method of treating diabetes melltus.
US5424394 discloses a solid phase peotide synthesis of amylin or amylin analogs using classical stepwise process wherein such synthetic human amylin is substantially free of deletion and other contaminating peptides having a purity to be at least about 95%. The invention also describes the use of Boc protection and double couplings cycles to get enhanced yield.
Amylin (37- mer peptide) contains a disulphide bridge from Cys-2 to Cys-7 and has an amidated C-terminus. During synthesis amylin is prone to aggregation because of excessive hydrophobic tendencies. This causes low coupling yields, incomplete couplings and side products. The addition of pseudoproline dipeptides to the peptide increases the purity of difficult peptides by decreasing the aggregation.
Amylin is a 37-mer amino acid peptide normone that is produced in the pancreas and co-secreted with insulin in response to serum glucose levels. Pramlintide is a synthetic analog of amylin that retains the biological activity of the hormone while offering superior physical and chemical properties that facilitate drug synthesis and development of a stable drug product for parenteral administration. Pramlintide is approved by US FDA for treating people with type 1 and type 2 diabetes.


The amino acid sequence for pramlintide is as follows:
There is one disulfide bridge between Cys 2 and Cys 7 and the underlined amino acids indicate the potential sites of deamidation at Asn, GIn and C-terminal tyrosine. Also the pramlintide sequence contains no free carboxyl groups, even at the amidated C-terminus (tyrosine). All the carboxyl groups in pramlintide are amidated, rendering the molecule cationic (protonated lysine, histidine, and arginine) at acidic pH. Pramlintide is isolated as a salt with acetate as the counterion. As with any drug substance, identifying and quantitating low levels of related substance impurities present in synthesis lots warrants a robust manufacturing process for pramlintide. Missed couplings, double-couplings, and L- to D- amino acid isomerizations are common errors that occur in peptide synthesis to yield, respectively, single-point amino acid deletion peptides, addition peptides, and diastereomers as related substances of the desired molecule. Specifically for pramlintide, the 37 amino acid length severely complicates detecting single amino acid modifications and requires highly selective analytical test methods for purity determinations. Degradation pathways for peptides in acidic aqueous solution frequently involve deamidation at asparagine and glutamine plus hydrolytic backbone cleavage. The 37- amino acid length and the 8 potential deamidation sites create a potential for many degradation products that differ from pramlintide by modificatons at only a single amino acid. Hence the synthesis of pramlintide poses significant challenge for manufacturers being a difficult peptide to synthesize.

Mutter et al.,1996 were the first to use Serine-, threonine-, and cysteine-derived cyclic building blocks (pseudo-prolines) to serve as reversible protecting groups for Ser, Thr, and Cys which have proven to be versatile tools for overcoming some intrinsic problems in the field of peptide chemistry. Multitude of peptides have been synthesized using Solid Phase Peptide Synthesis (SPSS). Subsequently, many efforts have been devoted to improving specific protecting groups, support systems, activation methods, and the automation of protocols. However, successful peptide assembly is still hampered by inherent problems such as poor solvation of the growing peptide chain during solid phase synthesis as well as limited solubility of fully protected peptide fragments in the solution approach, often leading to incomplete coupling steps. These undesirable physicochemical problems originate from intermolecular hydrophobic aggregation of the protected peptide chains and/or the formation of secondary structures, most notable p-sheets. Reported attempts to suppress the degenerative effect of such associations during aminoacylation reactions involve essentially "external factors" like solvent composition, elevated temperature, and use of chaotropic salts or solubilizing protecting groups which have been shown to have variable efficiencies. Hydrogen-bonded association has also been prevented by the introduction of an amide protecting group within the peptide chain.
Mutter et al., were the first to report that Ser/Thr-derived oxazolidine and Cys-derived thiazolidine derivatives exert a pronounced effect upon backbone conformation due to their structural similarity with proline itself. Due to the induction of a kink conformation in the peptide backbone, originating in the preference for cis amide bond formation, Tpro prevent peptide aggregation, self-association, and P-strucrure formation, thus improving the solvation and coupling kinetics of the growing peptide

chain considerably. These building blocks are readily accessible by cyclization of Ser, Thr, or Cys with aldehydes or ketones and serve as reversible protecting group in peptide synthesis. As a particular feature, variation of the C-2 substituents directly affects the ring stability, thus allowing for differential chemical stabilities in a variety of synthetic strategies.
Pseudoprolines play a crucial role in stepwise synthesis of Thr-, Ser-, or Cys-containing peptides, ψpro have the potential to simplify the purification of peptide fragment intermediates and to enhance the segment coupling kinetics when applied to segment condensation approach by SPPS. The synthesis of fully protected peptide is achieved by selective cleavage of the protected peptide fragment from the super acid labile resin whereas under such mild conditions (20% acetic acid), the C-2 substituted ψpro blocks (e.g.2,2-dimethyloxazolidine-4-carboxylic acid or 2-(2,4-dimethoxyphenyl)thiazolidine-4-carboxylic acid residues) remain stable. In the chemoselective litigation technique, it is desirable to preserve the ψpro ring system intact while the other amino acid side chains are deprotected. Alternatively, by concomitant cleavage of the acid labile protecting group, the resin anchor and the ψpro ring system, a fully deprotected peptide can be obtained in one single step. To summarise the general features of the ψpro concept in peptide synthesis can be derived: 1) ψpro represent a convenient, cheap, and easily accessible temporary protection technique for Ser, Thr, and Cys of comparable chemical stability as tBu or Trt side chain protecting groups; 2) ψpro building blocks are compatible with common strategies for peptide synthesis; 3) ψpro are introduced in peptides as N-protected dipeptides following the same protocols as conventional Fmoc-amino acids (e.g.HOBt/DIC) without racemization, essentially because of their C-terminal proline like structure; 4) the coupling of dipeptides or fragments containing C-terminal ψpro residues proceeds without racemization, offering a new tool in the convergent

synthesis of peptide fragments exhibiting C-terminal Ser, Thr, or Cys; 5) ψPro disrupt secondary structures (most notably p-sheets) and increase the solvation of the growing peptide chain during solid phase assembly.
WO93/10146 discloses solid phase peptide synthesis of 25 28 29Pro-h-Amylin using methylbenzhydrylamine anchor-bond resin and N-Boc/benzyl-side chain protection was carried out by standard peptide synthesis methods. The 2,7-[disulfide]amylin-MBHA-resin was obtained by treatment of Acm-protected cysteines with thallium (III) trifluoroacetate in trifluoroacetic acid. After cyclization was achieved the resin and side chain protecting groups were cleaved with liquid HF in the presence of dimethylsulfide and anisole. The 25,28 29Pro-h-Amylin was purified by preparative RP-HPLC. The peptide was found to be homogeneous by analytical HPLC and capillary electrophoresis and the structure confirmed by amino acid analysis and sequence analysis. The product gave the desired mass ionization FAB mass spec; (M + H)+ = 3,949.
An alternative strategy for assembling peptides is convergent synthesis. The challenge of convergent synthesis is to find suitable fragments and their coupling order for overcoming the known drawbacks of convergent synthesis. These drawbacks are solubility problems during coupling and purification, lower reaction rates compared to SPPS and a much higher racemization risk of the C terminal fragment during coupling. Pramlintide consists of thirty-seven amino acid residues so that a huge number of possible fragments and coupling order exists.
The linear, solid-phase synthesis of full length or partial peptides comprising this core portion encounters huge problems of individual coupling steps being utterly inefficient up to the point of near-impossibility even before repeated coupling.

Extending coupling times, raising coupling temperature etc. entail risk of increased racemisation, or undesired side products.
Aggregation and hence problems in solid phase synthesis is commonly believed in the art to correlate with occurrence of extended regions of β-sheet structure. A low β-sheet structure contents is common to most peptides of at least 10 amino acids length and do not correlate with any unusual problem in synthetic methodology.
Fmoc pseudoproline dipeptide units are nowadays commercially available; their synthesis has been described (Ruckle et al., Tetrahedron 1999, 55(37): 11281-11288; Keller et al., 1998, J. Am. Chem. Soc. 120:2714-2720). Pseudoprolines (ψR'R' pro) are oxazolidine or thiazolidine derivatives of serine, threonine, and cysteine residues that form on cyclocondensation of the amino acid with an aldehyde or ketone. Their five membered ring structure is reminiscent of a proline residue. These modified amino acid were introduced by Mutter and coworkers as temporary protecting groups for peptide synthesis and were found to exert a pronounced effect on the peptide backbone conformation. The oxazolidine derivatives are now extensively used as tools to impart better solubility to a growing peptide chain in solid-phase synthesis because they prevent aggregation of growing peptide chains and have been found to significantly increase the yields of difficult peptide sequences by improving solvation and peptide coupling kinetics. This is attributed to a conformational preference for cis-amide bond formation. The ability of pro residues to induce cis-amide bonds is well established by experimental and theoretical studies. In particular, disubstitution at the 2-C position (e.g. pro) strongly favours the cis-conformer
and can be used to tailor peptide backbone conformation for specific applications

Prior art teaches the use of Mutter's serine and Threonine derived Pseudoproline dipeptides for overcoming the synthesis challenges associated with difficult sequences and long peptides such as a low solvation during solid phase peptide synthesis and limited solubility of (un)protected peptide segments. Today a large number of pseudoproline building blocks are commercially available and their increasing popularity as value added protecting groups is based on advantages like: I) the insertion of ψ proline building blocks saves a coupling step and improves the solid phase peptide synthesis process which in turn facilitates and streamlines the HPLC purification protocols.
Pseudoproline dipeptides are basically incorporated in the most common peptide synthesis strategies such as a) they can be coupled to growing peptide chains using standard procedures and coupling reagents, b) They are easily cleaved with standard TFA mixtures, c) They are compatible with synthesis strategies involving Fmoc and Z-amino acids.
In one of the paper published by Merck, it has been stated that by using one Pseudoproline in the synthesis of a 20 mer biotinylated peptide, purified yields went from 11 to 27 mg with purity from 88% to 100%. It has also shown that in the preparation of highly aggregated sequences, 10-fold increases in product yield have been achieved from insertion of a single pseudoproline. However, for longer peptides, the incorporation of several pseudoprolines at regular intervals throughout the sequence has been found to be particularly effective.
PCT publication WO2009003666/ US20100249370 discloses the preparation of Pramlintide via a convergent three fragment synthesis strategy from the fragments

comprising the amino acid residues 1-12, 13-24 and 25-37 respectively. It specifies the use of three pseudoprolines moieties for the synthesis of Pramlintide, wherein it improves the solubility of the peptide and prevented or decreased it's aggregation. The convergent approach used synthesis of 1-12 fragment by inserting Fmoc-Ala-Thr (ψ Me,Me pro)- OH (pseudoproline at 9th position at Thr) after Gln(Trt)- on CTC resin with a loading of 0.64 mmole/g and 2.5 equivalent of each amino acid. TCTU/6-C1-HOBt/DIEA were used as coupling agents and 20% piperidine in NMP was used to deprotect the N-terminal Fmoc group. The peptide fragment was subjected to cyclization on the resin using 3 equivalents of iodine in DMF. 59% yield was reported. Subsequently, the second fragment 13-24 was synthesized by inserting Fmoc-Ser(tBu)-Ser(ψ Me,Me pro)-OH (pseudoproline at 20th position at Thr) after Asn (Trt)- on CTC resin with a loading rate of 0.66 mmol/g with 2.2-2.6 equivalent of each amino acid and 69% yield. The third fragment 25-37 was synthesized on Sieber resin by introducing Fmoc-GIy-Ser(ψ Me,Me pro)-OH (pseudoproline at 34th position at Ser) after Asn(Trt)- with loading rate of 0.55 mmole/g and yield of 45%. The three fragments are assembled, deprotected using TFA:TIS:Phe with recovery of 70% peptide and 97.5% purity after HPLC purification.
WO2009003666/ US20100249370 further discloses that several fragment coupling strategies consisting of: 1) synthesizing Boc-[l-24]-OH: A and H-[25-37]-NH2: B, coupling was difficult at positions 22, 21 and 9 and fragment A was not formed as detected by HPLC-MS; 2) synthesizing Boc-[1-20]-OH: A and H-[21-37]-NH2; B, coupling was difficult at positions 5 and 4 and fragment A was not formed as detected by HPLC-MS wherein the reaction conditions used HCTU/DIEA as coupling mixtures, use of Fmoc-19Ser-20Ser(ψ Me,Me pro)-OH (ψ Me,Me pro)-OH and cyclization after fragment coupling; 3) synthesizing Boc-[l-12]-OH: A and Fmoc-[13-20]-NH2:

B, and H-[21-37]NH2:C, coupling was difficult at positions 28,27 and 22 and precursor Fmoc-(C) was formed with only 11% purity as detected by HPLC wherein the reaction conditions used HCTU/DIEA as coupling mixtures, use of Fmoc-19Ser-20Ser(ψ Me,Me pro)-OH (ψ Me,Me pro)-OH and cyclization on resin with coupling order as (A) + (B) + (C). Thus the applicants claim to have surprisingly found a suitable strategy comprising the specific coupling of three peptide fragments, one of them containing the two cysteine residues with preformed disulfide bonds.
Page K. et al., 2007, discusses a fully automated fast Fmoc synthesis of human amylin 1-37 and on-resin disulfide bridge formation with pseudoproline dipeptides The incorporation of pseudoproline dipeptides into hAmylin1-13 improved the kinetics of its on-resin cyclization with Thallium (III) trifluoroacetate [TI (tfa)3]. Pseudoproline dipeptides do not require additional reaction time to couple and linear hAmylin 1-37 was synthesized in 8.5 hours as against 58 hours without using pseudoprolines. Sampson W.R. et al., undertook a comparative study between Hmb (2-hydroxy-4-methoxy-benzyl) protected amino acid and pseudoproline building block analogues for use in the solid phase synthesis of difficult peptides. Both of these derivatives act by blocking inter- and intramolecular hydrogen bonding, which has been shown to be a major cause of poor synthesis/quality/efficiency. While the two were shown to result in substantial improvements in the purity of crude peptides, pseudoproline incorporation was found to be superior to Hmb backbone protection. This was due to slow and incomplete coupling of the amino acid immediately following the Hmb amino acid.
Abedini et al., 2005 discloses efficient Fmoc solid phase peptide synthesis of the 37 residue human Amylin and its amyloidogenic 8-37 fragment using pseudorpoline

(oxazolidine) dipeptide derivative. Synthesis of hAmylin8-37 using Fmoc amino acids produced only traces of the desired peptide. Incorporation of pseudoproline dipeptides produced the desired product with high yield and allowed for the synthesis of the full length peptide. Three different strategies were used for the synthesis of hAmylin8-37. A) double coupling of the p-branched residues and those directly following P-branched residues, using only Fmoc protected amino acids; B) double coupling of all residues using only Fmoc protected amino acids; and C) incorporation of three oxazolidine pseudoproline dipeptide derivatives , with double coupling of β-branched residues, pseudoproline dipeptide derivatives, and residues following either of these. Three pseudoproline dipeptide derivatives were chosen for these synthesis, since previous studies have demonstrated that Fmoc-Xaa-Yaa-ψMe,Me pro units have only a local effect and that multiple use of these units leads to enhanced coupling yields throughout a difficult peptide synthesis. The first derivative, Fmoc-Ala-Thr(ψ Me,Me pro)-OH 1, was substituted for residues Ala-8 and Thr-9. The second derivative, Fmoc-Ser-Ser((ψ Me,Me pro)-OH 2, was inserted in place of Ser 19 and Ser 20; and the third derivative, Fmoc-Leu-Ser(ψ Me,Me pro)-OH 3, replaced residues Leu-27 and Ser-28.
Strategy A) failed, resulting in a crude analytical HPLC trace with multiple overlapping peaks, upon characterization by MALDI-TOF revealed multiple fragments of target peptide sequence eluting simultaneously and separation of these deletion peptides by HPLC was not possible. Strategy B was plagued by poor coupling as detected by the UV traces, but produced desired sequence after two failed attempts. The analytical HPLC trace of the crude material indicated several overlapping peaks which upon characterization by MALDI-TOF MS revealed three main products: the first product was the correct peptide [m/z =3226], the second correspond to a deletion peptide with a mass 112 Da too low, and the third was a

deletion peptide with a mass 114 Da too low. Furthermore, the correct peptide eluted at the same gradient positions at the two deletion peptides. The high level of impurities produced by strategy B also resulted in very poor solubility of the crude material, increasing the difficulty of purification by HPLC.
Strategy C was the most successful in producing the desired peptide with one major peak revealing the desired peptide [m/z= 3226], The high purity of the product dramatically increased the solubility of the crude material, making purification by HPLC very simple and effective. Following Strategy C, human Amylin 1-37 was synthesized. The high purity of the hAmylin 1-37 crude product allowed the direct oxidation to the Cys-2 to Cys-7 disulfide without purification.
White et al.,2004 describes the step-wise Fmoc solid phase synthesis of a 95- residue peptide related to FAS death domain. Attempts to prepare this peptide employing conventional amino acid building blocks failed. However, by the judicious use of dimethyloxazolidine dipeptides of serine and threonine, the peptide could be readily prepared in remarkable purity by applying single lh coupling reactions. The most striking results the author found were the consistency of the peak heights throughout the later stages of the assembly which is against the routine finding that during the assembly of long peptides the heights of the Fmoc deblock peaks gradually reduced as the synthesis progressed.
Yves-Marie Coic et al., 2010 contributes to highlight the benefits of pseudoproline dipeptide introductions in difficult SPPS. The author shows that a slight modification in the positioning choice conditioned the synthesis achievement of a 54 amino acid long Caveolin-1 peptide encompassing the intramembrane domain. The peptide was synthesized using conventional solid phase Fmoc/tert-butyl (tBu) chemistry with a 433A peptide synthesizer on a lOOumole scale. A low loading {0.23 mmole/g) PEG-polystyrene based NovaSyn R TGR resin as solid support. The resin is derivatised

with a modified Rink linker, which generates the carboxamide form of the peptide when subjected to TFA cleavage. HATU/D1PEA were used as coupling solvents and NMP washing between the piperidine deliveries and after coupling steps was introduced. New derivatives with eightfold molar excess with regard to resin were used and no postcoupling capping step with acetic anhydride was carried out. Pseudoproline dipeptides were introduced at Serl23, Serl04, and Thr95 positions. A competitive side reaction occurred during the coupling step, leading to a +42 adduct. The ratio of +42 fragment appeared to reflect the coupling difficulty and therefore the aggregation state of the chain. In order to improve the coupling yield of Ser88 and the difficulty to separate the target peptide from the truncated peptide, the pseudoproline dipeptide was incorporated at Thr93 position instead of Val-Thr(ψ Me,Me pro)- at Thr95.The crude peptide after synthesis and cleavage was entirely recovered by precipitation in diethylether. Thus by slightly modifying the pseudoproline dipeptide incorporation position might have favourably shifted the structure-disrupting effect so that improved Ser88 coupling kinetic prevented the capping side reaction.
Fayna Garcia-Martin et al.,2006, report an efficient stepwise solid-phase synthesis of RANTES (1-68) by combining the advantages of the totally PEG-based ChemMatrixR resin and pseudoproline dipeptides.
Valente AP et al.,2005, carried out HRMAS-NMR spectroscopy to examine the dynamics of an aggregating peptide sequences attached to a resin core with varying peptide loading (upto 80%) and solvent system. Strong peptide chain aggregation was observed mainly in highly peptide loaded resins when solvated in CDC13. Conversely, due to the better swelling of these highly loaded resins in DMSO, improved NMR

spectra were acquired in this polar aprotic solvent, thus enabling the detection of relevant sequence-dependent conformational alterations.
None of the prior art teaches, suggests or motivates use of mere single pseudoproline for the synthesis of difficult peptide like pramlintide.
It is an object of the present invention to provide a more efficient synthesis of pramlintide that overcomes the known drawbacks of solid phase peptide synthesis and is suitable for the production on an industrial scale. The object has been achieved by the synthesis according to Claim 1 and the peptide fragments of Claims 7 to 8.
OBJECT OF INVENTION:
It is an object of the present invention to provide a more efficient synthesis of pramlintide that overcomes the known drawbacks of solid phase peptide synthesis and is suitable for the production on an industrial scale. The object has been achieved by the synthesis according to Claim 1 and the peptide fragments of Claims 7 to 8.
One of the object of the present invention is the use of a single pseudoproline moiety for the synthesis of a 37-mer peptide, Pramlintide.
More particularly, the object of the present invention is a process for the production of pramlintide of formula 1


comprising:
a) coupling Fmoc-Tyr(X)-OH to acid labile solid support with substitution value
ranging from 0.15-0.60mmol/g, preferably about 0.25-0.35 mmol/g.
b) deprotecting the Fmoc protecting group using piperidine in 1
hydroxybenzotriazole (HOBt) in polar aprotic solvent;
c) assembling the Fmoc protected amino acids sequentially to yield peptide
fragment (21-37) of the formula 2
Fmoc-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin;
d) coupling Fmoc-Ser(X)-Ser(ψ Me,Me pro)-OH to N-terminal end of peptide
fragment of Formula 2 to yield peptide fragment of Formula 3:
Fmoc-Ser(X)-Ser(ψ Me,Me pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin
e) further coupling Fmoc-AA-OH to the peptide of formula 3 to yield peptide of
formula 4:
Fmoc-Lys(Boc)-Cys(Y)-Asn(Y)-Thr(X)-Ala-Thr(X)-Cys(Y)-Ala-Thr(X)-Gln(Y)-Arg(Pbf)-Leu-Ala-Asn(Y)-Phe-Leu-Val-His(Y)-Ser(X)-Ser(ψ Me,Me Pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin with a purity of crude peptide of > 30%; f) deprotecting and cleaving the peptide of Formula 4 from the resin, purifying the crude linear peptide by RP-HPLC and further oxidizing the linear peptide to cyclic peptide of Formula 1.

Still another object of the present invention is a peptide of Formula 4: Fmoc-Lys(Boc)-Cys(Y)-Asn(Y)-Thr(X)-Ala-Thr(X)-Cys(Y)-Ala-Thr(X)-Gln(Y)-Arg(Pbf)-Leu-Ala-Asn(Y)-Phe-Leu-Val-His(Y)-Ser(X)-Ser(ψ Me,Me pro)--Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin
Still another object of the present invention is a peptide of Formula 5: Fmoc-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(rBu)-Ala-Thr(tBu)-Cys(Trt)-Ala-Thr(rBu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-Ser(tBu)-Ser(ψ Me,Me pro)-Asn(rBu)-Asn{Trt)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr (rBu)-Tyr (tBu)-Resin.
SUMMARY OF THE INVENTION:
One aspect of the present invention is a process for the production of pramlintide of formula 1

comprising:
a) coupling Fmoc-Tyr(X)-OH to acid labile solid support with substitution value ranging from 0.15-0.60mmol/g, preferably about 0.25-0.35 mmol/g.
b) deprotecting the Fmoc protecting group using piperidine in 1 hydroxybenzotriazole (HOBt) in polar aprotic solvent;
c) assembling the Fmoc protected amino acids sequentially to yield peptide
fragment (21-37) of the formula 2

Fmoc-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn 30%;
f) deprotecting and cleaving the peptide of Formula 4 from the resin, purifying
the crude linear peptide by RP-HPLC and further oxidizing the linear peptide
to cyclic peptide of Formula 1.
Another aspect of the present invention is a peptide of Formula 4: Fmoc-Lys(Boc)-Cys(Y)-Asn(Y)-Thr(X)-Ala-Thr(X)-Cys{Y)-Ala-Thr(X)-Gln(Y)-Arg(Pbf)-Leu-Ala-Asn(Y)-Phe-Leu-Val-His(Y)-Ser(X)-Ser(ψ Me,Me pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin

Still another aspect of the present invention is a peptide of Formula 5: Fmoc-Lys(Boc)-Cys(Trt)-Asn(Trt)-Tnr(tBu)-AIa-Thr(tBu)-Cys(Trt)-AIa-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn (Trt)-Phe-Leu-Val-His(Trt)- Ser (tBu)- Ser (ψ Me,Me pro)-Asn(tBu)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-pro-pro-Thr(tBu)-Asn(Trt)-Val-Gly-
Ser(tBu)-Asn(Trt)-Thr (fBu)-Tyr (tBu)-Resin
BRIEF DECREPTION OF THE DRAWINGS:
Figure 1: RP-HPLC chromatogram of crude cleavage mixture of Pramlintide (Linear, non-cyclized) synthesized using hight loading Fmoc-Rink amide AM resin
Figure 2: RP-HPLC chromatogram of crude cleavage mixture of Pramlintide (Linear, non-cyclized) synthesized using low loading Fmoc-Rink amide AM resin
Figure 3: RP-HPLC chromatogram of crude cleavage mixture of Pramlintide (Linear, non-cyclized) (RP-HPLC purity 25%) synthesized using low loading Fmoc-Rink amide AM resin and single pseudoproline (Ser-Ser) 19-20
Figure 4: RP-HPLC chromatogram of crude cleavage mixture of Pramlintide (Linear, non-cyclized) (RP-HPLC purity 30%) synthesized using low loading Fmoc-Rink amide AM resin and using pseudoprolines (dipeptides) at two positions (Ser-Ser) 19-20 and (Thr-Asn) 3-4-
Figure 5 RP-HPLC chromatogram of crude cleavage mixture of Pramlintide (Linear, non-cyclized)(RP-HPLC purity 37%) synthesized using low loading Fmoc-Rink amide AM resin, using pseudoproline-(Ser-Ser) 19-20 and using 0.1M HOBt-piperidine-DMF combination
Figure 6 RP-HPLC chromatogram of crude cleavage mixture of Pramlintide (Linear, non-cyclized)(RP-HPLC purity 39%) synthesized using low loading Fmoc-Rink amide AM resin, using pseudoprolines at two positions (Ser-Ser) 19-20 and (Thr-Asn) 3-4 and using 0.lM HOBt-piperidine-DMF combination

Figure 7: RP-HPLC chromatogram of purified Pramlintide acetate (RP-HPLC purity
98.79 %) Figure 8: ESI-MS of Pramlintide acetate : Expected mass 3949 Da ; Mass Found:
3949.2 Da DETAILED DESCRIPTION OF THE INVENTION:
It is an object of the present invention to provide a more efficient synthesis of pramlintide that overcomes the known drawbacks of solid phase peptide synthesis and is suitable for the production on an industrial scale. The object has been achieved by the synthesis according to Claim 1 and the peptide fragments of Claims 7 to 8.
After several, unsuccessful experiments of using different strategies by varying loading substitution rates of the resin, by use of modified coupling agents like HBTU/NMM for synthesizing 37 mer amyloid analog in single stretch on a solid support and by using excess molar equivalent of Fmoc protected amino acids, the synthesis never progressed to completion. Prior art specifies the use of three pseudoproline moieties for the synthesis of full length hamylinl-37 which adds to the cost of synthesis. However, the applicants have surprisingly found that by just insertion of single pseudoproline moeity, the target crude peptide was synthesized with a remarkable purity of > 30%. Another surprising finding was by just adding 0.1M HOBt to the deprotection solvent, the purity of the crude peptide increased to > 35%.
One of the embodiment of the present invention is the use of a single pseudoproline moiety for the synthesis of a 37-mer peptide, Pramlintide.

Another embodiment of the present invention is a process for the production of pramlintide of formula 1

comprising:
a) coupling Fmoc-Tyr(X)-OH to acid labile solid support with substitution value ranging from 0.15-0.60mmol/g, preferably about 0.25-0.35 mmol/g.
b) deprotecting the Fmoc protecting group using piperidine in 1 hydroxybenzotriazole (HOBt) in polar aprotic solvent;
c) assembling the Fmoc protected amino acids sequentially to yield peptide fragment (21-37) of the formula 2
Fmoc-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin;
d) coupling Fmoc-Ser(X)-Ser(ψ Me,Me pro)-OH to N-terminal end of peptide fragment of Formula 2 to yield peptide fragment of Formula 3: Fmoc-Ser(X)-Ser(ψ Me,Me pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro- Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin
e) further coupling Fmoc-AA-OH to the peptide of formula 3 to yield peptide of formula 4:
Fmoc-Lys(Boc)-Cys(Y)-Asn(Y)-Thr(X)-Ala-Thr(X)-Cys(Y)-Ala-Thr(X)-Gln(Y)-Arg(Pbf)-Leu-Ala-Asn(Y)-Phe-Leu-Val-His(Y)-Ser(X)-Ser(ψ Me,Me Pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin
with a purity of crude peptide of > 30%;

f) deprotecting and cleaving the peptide of Formula 4 from the resin, purifying the crude linear peptide by RP-HPLC and further oxidizing the linear peptide to cyclic peptide of Formula 1.
Still another embodiment of the present invention is a peptide of Formula 4: Fmoc-Lys(Boc)-Cys(Y)-Asn(Y)-Thr(X)-Ala-Thr(X)-Cys(Y)-Ala-Thr(X)-Gln(Y)-Arg(Pbf)-Leu-Ala-Asn(Y)-Phe-Leu-Val-His(Y)-Ser(X)-Ser(ψ Me,Me pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thrp(X)-Asn(Y)-Val-Gly-Ser(X)-Asn{Y)-Thr (X)-Tyr (X)-Resin
Still another embodiemnt of the present invention is a peptide of Formula 5: Fmoc-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Ala-Thr(rBu)-Cys(Trt)-Ala-Thr(rBu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-Ser(?Bu)-Ser(ψ Me,Me pro)-Asn(tBu)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr (tBu)-Tyr (tBu)-Resin.
Yet another embodiment of the present invention is a pharmaceutical composition comprising peptide of Formula 1 manufactured by process of Claim 1 and pharmaceutically acceptable excipients.
Additional advantages of the invention will be set forth by the description which follows, and in part will be obvious from the description, or may be learnt by the practice of the invention. The advantages of the invention may be realised and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that the foregoing general description and the

following detailed description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
ABBREVIATIONS:
HOBt: N-hydroxy benzotriazole
DIC: Diisopropyllcarbodiimide
N-Boc: N-tert-butoxycarbonyl
MBHA resin: Methylbenzylhydrylamine resin
Acm: S-acetamidomethyl
HF: Hydrofluoric acid
TFA: Trifluroacetic acid
Fmoc: Fluorenylmethoxycarbonyl chloride
Z:Benzyl carbamate TCTU:O-(6-Chloro-1-hydrocibenzotriazol-1-yl)--1,1,3,3-tetramethyluronium
tetrafluoroborate 6-C1 HOBt:6-Chloro-1 -Hydroxy-1 H-Benzotriazole DIPEA: Diisopropylethylamine NMP: N-methylpyrrolidone DMF: Dimethyl formamide CTC resin: Chlorotrityl resin TIS: Tri isopropylsilane DTT: Dithiothreitol EDT: Ethylenediamine tetraceticacid Hmb: (2-hydroxy-4-methoxybenzyl) HATU:2-(1H-7Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronoum
hexafluorophosphate Methanaminium CDC13:Deuterated Chloroform

DMSO: Dimethyl Sulfoxide
EXAMPLES:
Synthesis of linear precursors on solid support using Pscudoproline (19Fmoc-Ser(tBu)-20Ser(ψ Me,Me pro)-OH):
Fmoc-Rink amide AM resin with a low substitution value ranging from 0.28-0.33 mmole/gram (about 8.3 gm resin, 2.5 mmole) was swelled in DMF for 1 hr by agitation under nitrogen. The Fmoc protecting group was removed by treating with 20% piperidine in DMF, followed by the through washing of the resin with DMF. The coupling of the first amino acid Fmoc-Tyr(tBu)-OH (6.25-8.75 mmole, 2.5-3.5 eq)), was carried out by addition of HBTU (6.25-8.75 mmole, 2.5-3.5 eq) and NMM (0.4M in DMF). The mixture was stirred for 1 hr. The completion of coupling reaction, was confirmed by the ninhydrine test after washing of the resin. The subsequent removal of the Fmoc protecting group was removed with 20% piperidine in DMF/ 20% piperidine in 0.1M HOBt in DMF in all the amino acid deprotections. The resin was thoroughly washed with DMF before the addition of the next amino acid. These steps were repeated each time with the successive amino acid according to the peptide sequence. The trifunctional amino acid were side chain protected as follows: Arg(pbf), Asn(trt), Tyr(tBu), Cys(Trt), Lys(Boc), Thr(tBu), Ser(tBu), Gln(Trt), His(Trt). Post completion of the synthesis, the resin was thoroughly washed with DMF and DCM. The pseudoproline dipeptide, Fmoc-Ser(tBu)-Ser(ψ Me,Me pro)-OH) was incorporated using the standard synthesis cycle using 2.5X excess only. Additionally pseudoproline dipeptide, Fmoc-Thr(tBu)-Asn((ψ Me,Me pro)-OH) was incorporated using the standard synthesis cycle using 2.5X excess only at position 3 and 4 of the 37mer peptide.

Weight of the peptidyl resin: 22-25gm
Cleavage of Linear Precursors on Solid Support:
The cleavage of the peptide from the resin with simultaneous deprotection of all the protecting groups was carried out by the treatment of either of the cocktail cleavage mixtures stated below:
1. TFA: TIS: Water (95:2.5:2.5 v/v)
2. TFA:TIS:DTT:Water (94:01:2.5:2.5 v/v;w/v)
3. TFA: TIS: Phenol: DTT: Water (82.5:05:0.5:2.5:05 v/v; w/v)
4. TFA: DTT: Thioanisol: Phenol: Water (82.5:2.5:05:05:05 v/v; w/v)
5. TFA: EDT: Thioanisol: Phenol: Water (82.5: 2.5: 05: 05: 05 v/v)
6. TFA: TIS: Water: EDT (94: 01: 2.5: 2.5 v/v)
The cleavage was carried out at 2-8°C for 20 minutes followed by then stirring the peptidyl resin for 2.5 hours at ambient temperature. The crude cleavage mixture was then filtered, the resin washed thoroughly with TFA. The crude cleavage peptide solution was concentrated on a rotary evaporator. The precipitation was effected using cold diisopropyl ether (DIPE) and_stored at -20°C overnight. The peptide precipitate was filtered and dried under vaccum for 16 hrs. The preferred cleavage mixture was the K reagent replacing EDT with DTT of them all.
The isolated yield of the crude peptide:.6-8g.

Synthesis of linear precursors on solid support using low loading Fmoc-Rink amide AM resin (0.28-0.333mmol/g):
Fmoc-Rink amide AM resin with a low substitution value ranging from 0.28-0.33 mmole/gram (about 8.3 gm resin, 2.5 mmole) was swelled in DMF for 1 hr by agitation under nitrogen. The Fmoc protecting group was removed by treating with 20% piperidine in DMF, followed by the through washing of the resin with DMF. The coupling of the first amino acid Fmoc-Tyr(tBu)-OH (6.25-8.75 mmole, 2.5-3.5 eq)), was carried out by addition of HBTU (6.25-8.75 mmole, 2.5-3.5 eq)) and NMM (0.4M in DMF). The mixture was stirred for 1 hr. The completion of coupling reaction, was confirmed by the ninhydrine test after washing of the resin. The subsequent removal of the Fmoc protecting group was removed with 20% piperidine in DMF in all the amino acid deprotections. The resin was thoroughly washed with DMF before the addition of the next amino acid. These steps were repeated each time with the successive amino acid according to the peptide sequence. The trifunctional amino acid were side chain protected as follows: Arg(pbf), Asn(trt), Glu(OtBu), Tyr(tBu), Cys(Trt), Lys(Boc), Thr(tBu), Ser(tBu), Gln(Trt), His(Trt). Post completion of the synthesis, the resin was thoroughly washed with DMF and DCM. The crude purity of the product was highly inconsistent and in some of the batches the product was not formed or was of unacceptable quality (Figure 2).
Similarly synthesis of pramlintide was carried out using standard loading range of 0.50-0.60 mmole/g. Performing the synthesis using this strategy, the crude purity of the product was highly inconsistent and in some of the batches the product was not formed or was of unacceptable quality (Figure 1).

Cleavage of Linear Precursors on Solid Support:
The cleavage of the peptide from the resin with simultaneous deprotection of all the protecting groups was carried out by the treatment of either of the cocktail cleavage mixtures stated below:
l.TFA: TIS: Water(95:2.5:2.5 v/v)
2.TFA:TIS:DTT:Water(94:01:2.5:2.5 v/v; w/v)
3.TFA: TIS: Phenol: DTT: Water (82.5:05:0.5:2.5:05 v/v;w/v)
4.TFA: DTT: Thioanisol: Phenol: Water (82.5:2.5:05:05:05 v/v;w/v)
5.TFA: EDT: Thioanisol: Phenol: Water (82.5: 2.5: 05: 05: 05 v/v)
6.TFA: TIS: Water: EDT (94: 01: 2.5: 2.5 v/v)
The cleavage was carried out at 2-8cC for 20 minutes followed by then stirring the peptidyl resin for 2.5 hours at ambient temperature. The crude cleavage mixture was then filtered, the resin washed thoroughly with TFA. The crude cleavage peptide solution was concentrated on a rotary evaporator. The precipitation was effected using cold diisopropyl ether (DIPE) and_stored at -20°C overnight. The peptide precipitate was filtered and dried under vaccum for 16 hrs. The preferred cleavage mixture was the K reagent replacing EDT with DTT of them all.
The crude purity of the product using 20% piperidine in DMF and single pseudoproline was determined by HPLC (Figure 3). The crude purity of the product using 20% piperidine in DMF and two pseudoprolines was determined by HPLC

(Figure 4). The crude purity of the product using 20% piperidine in 0.1M HOBt in DMF and single pseudoproline was determined by HPLC (Figure 5).The crude purity of the product using 20% piperidine in 0.1M HOBt in DMF and two pseudoprolines was determined by HPLC (Figure 6).
Purification of the Linear Peptide:
The crude linear peptide was taken up for purification by RP-HPLC. Post purification the fractions were analyzed for the purity. The fraction containing purity in the range of 75-85% were pooled and lyophilized and were further taken up for the oxidation or were taken in the solution form itself.
Oxidation of the Linear Peptide:
a) Using Hydrogen peroxide oxidation method:
The peptide was dissolved in 20% acetic acid at concentration of 0.1- l.Omg/ml, followed by the addition of aqueous ammonia solution (28%) to adjust the pH to. about 8.5. Calculated fixed amount of Hydrogen peroxide solution was added at regular intervals. After the addition of peroxide ceased, the reaction mixture was stirred for about 1 hr-2hrs. The reaction was quenched by lowering the pH to 3.0 by using 20% acetic acid. The reaction mixture was then taken up for the RP-HPLC analysis & further purified
b) Using Copper sulfate oxidation method
The peptide was dissolved in 25% acetonitrile in 0.1% TFA at concentration of 1.0-2.0 mg/ml, followed by the addition of ammonium hydroxide solution to adjust the pH to about 8.5. Calculated amount of copper sulfate (solid or in water) was added.

The reaction mixture was stirred overnight, till the completion of oxidation, as monitored by RP-HPLC. The reaction was quenched by lowering the pH to 4.0 by using 20% acetic acid. The reaction mixture was then taken up for the RP-HPLC analysis & further purified
Alternatively the oxidation reactions can also be carried out by using standard reagents like Iodine-methanol, Iodine-acetic acid, or air oxidation using the optimized conditions.
Purification of the Cyclic Peptide & Salt exchange:
The cyclic peptide was taken up for purification by RP-HPLC. Post purification, the fractions were analyzed for the purity. The fraction containing >97% were pooled and lyophilized and were further taken up for salt exchange. The salt exchange was performed only RP-HPLC using acetate buffer or ammonium acetate. The fractions were pooled and were lyophilized and stored at -20°C.
The salt exchange was carried out by using ion exchange chromatography. Medias such as Dowex ion exchange resins could also be used for the salt exchange.
The overall isolated purification yield was 5-10%. HPLC chromatogram of purified pramlintide acetate is depicted in Figure 7 and mass is depicted in Figure 8.

We Claim,
1) A process for the production of pramlintide of Formula 1:

comprising:
a) coupling Fmoc-Tyr(X)-OH to acid labile solid support with substitution value ranging from 0.15-0.60mmol/g, preferably about 0.25-0.35 mmol/g;
b) deprotecting the Fmoc protecting group using piperidine in 1 hydroxybenzotriazole (HOBt) in polar aprotic solvent;
c) assembling the Fmoc protected amino acids sequentially to yield peptide fragment (21-37) of the formula 2
Fmoc-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin;
d) coupling Fmoc-Ser(X)-Ser(ψ Me,Me pro)-OH to N-terminal end of peptide fragment of Formula 2 to yield peptide fragment of Formula 3: Fmoc-Ser(X)-Ser(ψ Me,Me pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-IIe-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn{Y)-Thr (X)-Tyr (X)-Resin
e) further coupling Fmoc-AA-OH to the peptide of formula 3 to yield peptide of formula 4:
Fmoc-Lys(Boc)-Cys(Y)-Asn(Y)-Thr(X)-Ala-Thr(X)-Cys(Y)-Ala-Thr(X)-Gln(Y)-Arg(Pbf)-Leu-Ala-Asn{Y)-Phe-Leu-Val-His(Y)-Ser(X)-Ser(ψ Me,Me

Pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr (X)-Tyr (X)-Resin with a purity of crude peptide of > 30%; f) deprotecting and cleaving the peptide of Formula 4 from the resin, purifying the crude linear peptide by RP-HPLC and further oxidizing the linear peptide to cyclic peptide of Formula 1.
2. The process of claim 1, wherein the acid labile solid support is selected from the group consisting of Rink amide AM resin, 5-(4-iV-Fmoc-aminomethyI-3,5 dimethoxyphenoxy)valeryl (PAL resin), Nova PEG Rink amide, NovaSyn TG R resin, Rink amide MBHA resin, Rink Amide NovaGel, Rink amide PEGA resin, preferably is Rink amide AM resin.
3. The process of claim 1, wherein the polar aprotic solvent used is dimethyl formamide(DMF).
4. The process of claim 1, wherein the trifunctional amino acids were side chain protected with X group selected from tBu, Trt, Chloro trityl preferably tBu and Y group selected from Trt, Tmob, Mtt, Xan preferably Trt.
5. The process of claim 1, wherein the deprotecting solvent comprises of 20%-50% piperidine in 0.1M -0.5M-hydroxybenzotriazole (HOBt) in DMF.
6. The process of claim 1, wherein the peptide of Formula 4 is subjected to air oxidation or hydrogen peroxide or copper sulphate or iodine oxidation to yield cyclic peptide of Formula 1.
7. A peptide of Formula 4:
Fmoc-Lys(Boc)-Cys(Y)-Asn(Y)-Thr(X)-Ala-Thr(X)-Cys(Y)-Ala-Thr(X)-GIn(Y)-Arg(Pbf)-Leu-AIa-Asn(Y)-Phe-Leu-Val-His(Y)-Ser(X)-Ser(ψ Me,Me Pro)-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr {X)-Tyr (X)-Resin

8. A peptide of Formula 5:
Fmoc-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Ala-Thr(rBu)-Cys(Trt)-Ala-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-Ser(tBu)-Ser(ψ Me,Me pro)-Asn(tBu)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(tBu)-Resin.
9. A composition comprising peptide of Formula 1 obtained by process of claim 1 and essentially pharmaceutically acceptable excipients.
10. Pramlintide and process for preparation thereof substantially as described herein with reference to the foregoing examples and/or accompanying drawings.